Condensed tannins (proanthocyanidins) are widespread in the plant kingdom, form part of the human diet, and display multiple biological activities that render them significant to health. Procyanidins have attracted a great deal of recent attention in the fields of nutrition, medicine and health due to their wide range of potentially significant biological activities. There is a growing body of evidence suggesting that these compounds act as potent antioxidants in vitro, ex vivo and in vivo and may thus alter the pathophysiology of imbalances or perturbations of free radical and/or oxidatively driven processes in many diseases or directly interfere with many cellular processes. See Nijveldt, R. J. et al., Am. J. Clin. Nutr. 2001, 74, 418. Initial observations also have shown that procyanidin-rich fractions extracted from defatted cocoa beans elicited in vitro growth inhibition in several human cancer cell lines. See U.S. Pat. No. 5,554,645 issued Sep. 10, 1996 to L. J. Romanczyk, Jr. et al., the disclosure of which is incorporated by reference.
Isolation, separation, purification, and identification methods have been established for the recovery of a range of procyanidin oligomers for comparative in vitro and in vivo assessment of biological activities, and currently some oligomers can be synthesized using time-consuming methods. For instance, previous attempts to couple monomeric units in free phenolic form using mineral acid as the catalyst in aqueous media have met with limited success. The yields were low, the reactions proceeded with poor selectivity, and the oligomers were difficult to isolate. See Steynberg, P. J., et al., Tetrahedron, 1998, 54, 8153–8158. An overview of the shortcomings is set out below.
The benzylated monomer was prepared from the free monomer using benzyl bromide in combination with potassium carbonate (K2CO3) and dimethyl formamide (DMF). See Kawamoto, H. et al., Mokuzai Gakkashi, 1991, 37, 741–747. The yield, however, was only about 40%. In addition, competing C-benzylation leads to a mixture of products, which make isolation of the benzyl-protected target monomer more difficult. Also, partial racemization of (+)-catechin at both the C-2 and C-3 positions was observed (see Pierre, M.-C. et al., Tetrahedron Letters, 1997, 38, 5639–5642).
Two primary methods for oxidative functionalization are taught in the literature. See Betts, M. J. et al., J. Chem. Soc., C, 1969, 1178 and Steenkamp, J. A., et al., Tetrahedron Lett., 1985, 3045–3048. In the older method, protected (+)-catechin was treated with lead tetraacetate (LTA) in benzene to produce the 4β-acetoxy derivative which was then successfully hydrolyzed to the 3,4-diol. Flavan-3,4-diols are incipient electrophiles in the biomimetic synthesis of procyanidins. The major drawback in the oxidative functionalization of the prochiral benzylic position was a low yield (30–36%) of the acetate during the lead tetraacetate (LTA) oxidation. The more recent method of oxidatively functionalizing the C-4 position relies on the use of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In this method, the protected monomer was treated with DDQ in methanol. This allows introduction of a methoxyl group at the C-4 position in a stereoselective manner. The yield was about 40–50%
There are a number of reports on the coupling reaction between monomers and their 3,4-diols in aqueous acid. These methods are unsatisfactory because of low yields, lack of selectivity, and difficulty in the purification from aqueous media. See Kawamoto, H. et al., J. Wood Chem. Technol., 1989, 9, 35–52 who report the titanium tetrachloride (TiCl4) mediated coupling between 4-hydroxy-tetra-O-benzyl-(+)-catechin and 5 equivalents (eq.) of tetra-O-benzyl-(+)-catechin to produce a 3:2 mixture of 4α,8 and 4β,8 catechin dimers. This coupling leads to the 4β,8-dimer together with higher oligomers in yields that decrease with the increasing molecular mass of the oligomer.
Using a 2,3-cis-3,4-trans-flavan-3,4-diol, procyanidins B2 and B5 derivatives were synthesized. The diol was prepared by the acyloxylation of the C-4 benzylic function of a (−)-epicatechin tetramethyl ether with lead tetraacetate in a benzene solution. This oxidative functionalization of the C-4 position of the methyl-protected epicatechin monomer was improved by using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ) in methanol to introduce a methoxyl group at the C-4 position. The protected C-4 methoxy monomer was used in the synthesis of 4,8-linked linear procyanidin oligomers up to the trimers. See Steenkamp et al., Tetrahedron. Lett. 1985, 26, 3045–3048.
Procyanidin oligomers were prepared using a protected epicatechin or catechin monomer having, as a C-4 substituent, a C2–C6 alkoxy group having a terminal hydroxy group such as a 2-hydroxyethoxy group. The protecting groups used are those that do not deactivate the A ring of the monomer, e.g., benzyl protecting groups. See Kozikowski, A. P. et al. J. Org. Chem. 2000, 65, 5371–5381 and U.S. Pat. No. 6,207,842 (issued Mar. 27, 2001 to Romanczyk, L. J. et al.). The C-4 derivatized, protected monomer was coupled with a protected catechin monomer or protected epicatechin monomer to form a protected 4,8-dimer which was then deprotected or used for further coupling with another protected, C-4 derivatized epicatechin monomer to form protected higher 4,8-oligomers. If a 4,6-linkage was desired, the C-8 position of the protected catechin or epicatechin monomer was blocked with a halogen group prior to coupling with the C-4 derivatized, protected epicatechin monomer or oligomer. Higher oligomers having both 4,8- and 4,6-linkages were also prepared. The protected dimers or oligomers were deprotected, and if necessary, deblocked. The coupling was carried out in the presence of a protic acid or a Lewis acid such as titanium tetrachloride (TiCl4). The stereochemical nature of the interflavan bond was confirmed by the synthesis of a specifically protected derivative and its subsequent degradation reference. Unfortunately, titanium tetrachloride-mediated further chain extension of the epicatechin dimer leads to the formation of regioisomers. This is a serious drawback, not only in terms of yield, but also purity. Even though the 4β,8-trimers and 4β,8-tetramers were isolated in pure form, the same cannot automatically be expected for the larger oligomers, for which the number of possible isomers, and thus contaminants, grows rapidly.
One potential way of dealing with this problem is to carefully purify the chain-extended oligomer after each step in order to ensure that all chain-extended oligomers are at least derived from a single isomer of the starting oligomer. However, upon the titanium tetrachloride-mediated chain extension of the protected trimer (2 eq.) with the C-4 derivatized, protected monomer, not only were the protected tetramer, pentamer, and small amounts of higher oligomers formed, but the protected trimer was degraded to the monomer and dimer, which then participated in the chain-extension reaction, giving rise to regioisomeric oligomers such as small amounts of the protected 4β,6:4β,8-trimer. While the reaction conditions (methylene chloride/tetrahydrofuran (9:11), 0° C., 15 min, then room temperature, 140 min) were not optimized, the observation of this deleterious aide reaction warranted a search for a better synthetic approach.
Thus, there is a need for improved methods for synthesizing epicatechin or catechin oligomers, particularly the higher oligomers, and a process for using protected larger epicatechin or catechin oligomers as building blocks for chain extension to even larger oligomers.